Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of Penaeus monodon in Pichia pastoris Supattra Treerattrakool, 1 Apinunt Udomkit, 1, * Lily Eurwilaichitr, 2 Burachai Sonthayanon, 1 and Sakol Panyim 1 1 Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand 2 BIOTEC Training Center of Genetic Engineering and Biotechnology at Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakhon Pathom 73170, Thailand Abstract: Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the a-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 lg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon. Key words: a-mating factor, arthropod, black tiger prawn, crustacean hyperglycemic hormone (CHH), Pichia pastoris, yeast expression system. INTRODUCTION The X-organ sinus gland (XOSG) complex, located in the optic ganglia in the eyestalk, is the major neuroendocrine control center of crustaceans. The major hormone family produced in the XOSG complex is composed of crustacean Recieved May 15, 2002; accepted September 5, 2002. *Corresponding author: telephone 66-2-4419003, ext. 1237, 1253; fax 66-2-4419906; e-mail staud@mahidol.ac.th Mar. Biotechnol. 5, 373–379, 2003 DOI: 10.1007/s10126-002-0078-x Ó 2003 Springer-Verlag New York Inc.